Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Bellevalia ciliata (Cyr.) Nees (Hyacinthaceae) neu für Bulgarien

(Bellevalia ciliata was recorded in north-east Bulgaria south of the Dobrudsha, within field and steppe vegetation. Vegetation records and a distribution map are presented. Based on taxonomic studies it is proposed to combineB. ciliata, B. sarmatica (Pall.) Wor. andB. speciosa Wor. under the oldest nameB. ciliata (Cyr.) Nees.     

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log₁₀ and 2.0 log₁₀ units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log₁₀ units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

Assessing the impact of ozone on photosynthesis of European beech (Fergus sylvatica L.) in environmental chambers

An exposure — response study with proportionalto-ambient ozone levels was conducted in closed chambers on 3-year-old European beech (Fagus sylvatica L.) of montane origin. The fumigation started in April 1990 and lasted for a single growing season. Climate data and ozone concentrations monitored at an experimental station of the Institute for Applied Plant Biology, Schönenbuch, Switzerland were simulated in the exposure chambers 12 days later (1*O₃). To test exposure-response relations three additional treatments were applied, subambient (0.2*O₃) and two proportionally increased ozone treatments (1.5*O₃ and 2*O₃). The photosynthetic behaviour of the trees in August revealed the light reactions to be less affected than parameters which are related to the dark reactions of photosynthesis. Assimilation (A₃₅₀), apparent carboxylation efficiency (CE), and maximum photosynthetic capacity (A₂₅₀₀) were reduced with increasing ozone concentration. For the ozone response of CE and A₂₅₀₀ Critical Levels were calculated.     

Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.     

Evidence for the Role of MAP1B in Axon Formation

Cultured neurons obtained from a hypomorphous MAP1B mutant mouse line display a selective and significant inhibition of axon formation that reflects a delay in axon outgrowth and a reduced rate of elongation. This phenomenon is paralleled by decreased microtubule formation and dynamics, which is dramatic at the distal axonal segment, as well as in growth cones, where the more recently assembled microtubule polymer normally predominates. These neurons also have aberrant growth cone formation and increased actin-based protrusive activity. Taken together, this study provides direct evidence showing that by promoting microtubule dynamics and regulating cytoskeletal organization MAP1B has a crucial role in axon formation.     

Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.     

Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase

The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.